Fish scale source zinc chelating peptide as well as preparation method and application thereof

Abstract

The invention relates to a fish scale source zinc chelating peptide. The amino acid sequence of a polypeptide part of the fish scale source zinc chelating peptide is HAR. Therefore, the polypeptide part of the fish scale source zinc chelating peptide has high zinc affinity, a nitrogen atom in a histidine imidazole group at the N terminal is coordinated with Zn < 2 + > through lone pair electrons, and meanwhile, a carboxyl oxygen atom of arginine at the C terminal can be used as an electron donor and is further coordinated with Zn < 2 + >, so that the zinc chelating peptide is obtained. The obtained fish scale source zinc chelating peptide enables HAR which does not have antibacterial activity originally to show excellent broad-spectrum antibacterial activity and biological membrane dispersion effect. In addition, HAR is chelated with Zn < 2 + > through a coordinate bond, so that the stability of the polypeptide is remarkably improved, and the defect that a peptidyl antibacterial agent is easily hydrolyzed by protease is overcome. The fish scale source zinc chelating peptide provided by the invention can provide antibiotic substitutes for food and agriculture, prolong the shelf life of food, and promote the reutilization of fishery by-products.

Description

Technical Field

[0001] The present invention relates to the technical field of food microbiology, and particularly relates to a zinc-chelated peptide derived from fish scales, a preparation method thereof, and an application thereof. Background Art

[0002] Due to the abuse of antibiotics in the fields of medicine, food, etc., the problem of drug resistance of many pathogenic bacteria to antibiotics is serious. Therefore, it is urgent to find a new type of antibacterial agent that is safe, efficient and not easily cause drug resistance.

[0003] In recent years, the research on the formation of chelates between trace elements and bioactive peptides has attracted much attention. Zinc, as one of the essential trace elements for humans and animals, has important biological functions. When a bioactive peptide binds to a zinc ion to form a chelate, its biological activity can be effectively enhanced, such as antioxidant and antibacterial effects. At the same time, zinc-chelated peptides can, on the one hand, overcome the toxic side effects and non-persistent antibacterial properties caused by the easy precipitation and oxidation of inorganic zinc, and on the other hand, improve the stability of bioactive peptides. At present, the research on zinc-chelated peptides is still at a shallow level.

[0004] Fishery by-products such as fish scales, fish skins and bones are rich in proteins, but are often discarded due to their low economic value. At present, the structural research on zinc-chelated peptides derived from fish scales is not deep enough, and their biological activities have not been fully explored, especially in terms of zinc-chelated peptides derived from fish scales with broad-spectrum antibacterial activity.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a zinc-chelated peptide derived from fish scales, a preparation method thereof, and an application thereof.

[0007] The technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a zinc-chelated peptide derived from fish scales, and the amino acid sequence of its polypeptide part is HAR.

[0009] A zinc-chelated peptide derived from fish scales proposed by the present invention has high zinc affinity in its polypeptide part. The nitrogen atom in the imidazole group of histidine at the N-terminus coordinates with Zn2+ through a lone pair of electrons. At the same time, the carboxyl oxygen atom of arginine at the C-terminus can act as an electron donor to further coordinate with Zn2+. The obtained zinc-chelated peptide derived from fish scales enables HAR, which originally does not have antibacterial activity, to exhibit excellent broad-spectrum antibacterial activity and biofilm dispersion effect. In addition, HAR chelates with Zn2+ through a coordination bond, significantly improving the stability of the polypeptide and avoiding the deficiency that peptide-based antibacterial agents are easily hydrolyzed by proteases. The zinc-chelated peptide derived from fish scales of the present invention can provide an antibiotic substitute for food and agriculture, extend the shelf life of food, and promote the reuse of fishery by-products.

[0010] Second aspect, the present invention provides a method for preparing fish scale-derived zinc chelated peptide as described in the above technical solution, comprising the following steps: mixing a polypeptide aqueous solution and an inorganic zinc solution, adding an organic solvent for chelation reaction, washing the obtained product, centrifuging to collect the precipitate, and freeze-drying to obtain the fish scale-derived zinc chelated peptide HAR@Zn.

[0011] The preparation method adopted by the present invention has a simple process and can use fish scales as the source of green ligands for zinc chelated peptides, providing a technical reference for the intensive processing and high-value utilization of fish scales.

[0012] Preferably, the concentration of the polypeptide aqueous solution is 0.1 - 0.15 g / mL, the concentration of the inorganic zinc solution is 2 mol / L, and the mass ratio of the polypeptide to the inorganic zinc is 0.13 - 0.15:1.

[0013] Preferably, the temperature of the chelation reaction is 30 - 40 °C, and the chelation reaction time is 12 - 16 h.

[0014] Third aspect, the present invention provides an application of the fish scale-derived zinc chelated peptide as described in the above technical solution or the fish scale-derived zinc chelated peptide prepared by the preparation method as described in any one of the above technical solutions in the preparation of antibacterial products.

[0015] Preferably, the objects of action of the antibacterial product include Gram-negative bacteria and Gram-positive bacteria.

[0016] Preferably, the Gram-negative bacteria include Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, and Pseudomonas aeruginosa, and the Gram-positive bacteria include Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Enterococcus faecalis.

[0017] The fish scale-derived zinc chelated peptide of the present invention has broad-spectrum antibacterial activity, has good inhibitory effects on a variety of Gram-negative bacteria and Gram-positive bacteria, and particularly has inhibitory and eradication effects on the planktonic cells and biofilm strains of Vibrio alginolyticus 2512, and has broad application prospects in the inhibition of pathogenic bacteria. Description of the Drawings

[0018] Figure 1 For the structural characterization of the polypeptide and fish scale-derived zinc chelated peptide in Example 1, wherein, A is the secondary mass spectrum of polypeptide HAR, and B is the Fourier transform infrared spectrum of polypeptide HAR and fish scale-derived zinc chelated peptide HAR@Zn;

[0019] Figure 2 For the minimum inhibitory concentration (MIC) graph of fish scale-derived zinc chelated peptide HAR@Zn against Vibrio alginolyticus 2512 in Example 1;

[0020] Figure 3 To study the effect of the fish scale-derived zinc chelated peptide HAR@Zn in Example 1 on the anti-biofilm activity against Vibrio alginolyticus 2512;

[0021] Figure 4 To study the antibacterial spectrum of the fish scale-derived zinc chelated peptide HAR@Zn in Example 1;

[0022] Figure 5 To study the effect of the fish scale-derived zinc chelated peptide HAR@Zn in Example 1 on the hemolytic activity of mouse red blood cells;

[0023] Figure 6 To study the cytotoxicity of the fish scale-derived zinc chelated peptide HAR@Zn in Example 1 on mouse macrophage RAW 264.7;

[0024] Figure 7 To study the stability of the fish scale-derived zinc chelated peptide HAR@Zn in Example 1. Detailed implementation manners

[0025] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0026] On the one hand, the present invention provides a fish scale-derived zinc chelated peptide, and the amino acid sequence of its polypeptide part is HAR.

[0027] On the other hand, the present invention provides a preparation method of the fish scale-derived zinc chelated peptide as described in the above technical solution, including the following steps: mixing a polypeptide aqueous solution with an inorganic zinc solution, adding an organic solvent for chelation reaction, washing the obtained product, centrifuging to collect the precipitate, and freeze-drying to obtain the fish scale-derived zinc chelated peptide HAR@Zn.

[0028] In the embodiments of the present invention, the polypeptide can be purified from the hydrolyzate of Lutjanus erythropterus fish scale protein to obtain the peptide sequence and synthesized by solid-phase synthesis technology, which is not particularly limited herein. See the secondary mass spectrum of HAR in Figure 1 A. In addition, in the embodiments of the present invention, the organic solvent uses absolute ethanol, which can accelerate the capture of Zn2+ by the polypeptide; the inorganic zinc uses ZnSO4·7H2O.

[0029] In a preferred embodiment of the present invention, the concentration of the polypeptide aqueous solution is 0.1-0.15 g / mL, the concentration of the inorganic zinc solution is 2 mol / L, and the mass ratio of the polypeptide to the inorganic zinc is 0.13-0.15:1.

[0030] In a preferred embodiment of the present invention, the temperature of the chelation reaction is 30 to 40 °C, and the chelation reaction time is 12 to 16 h.

[0031] The present invention further provides an application of a fish scale-derived zinc chelated peptide according to the above technical solution or a fish scale-derived zinc chelated peptide prepared by the preparation method according to any one of the above technical solutions in the preparation of antibacterial products.

[0032] In a preferred embodiment of the present invention, the objects of action of the antibacterial product include Gram-negative bacteria and Gram-positive bacteria.

[0033] In a preferred embodiment of the present invention, the Gram-negative bacteria include Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacteria include Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus and Enterococcus faecalis.

[0034] Example 1

[0035] A preparation method of a fish scale-derived zinc chelated peptide includes the following steps:

[0036] Mix an aqueous polypeptide solution (1.21 g of polypeptide HAR dissolved in 10 mL of ultrapure water) with an inorganic zinc solution (15 mL, 2 mol / L ZnSO4·7H2O), add anhydrous ethanol with a volume 2.5 times that of the mixed solution for chelation reaction, react at 35 °C for 16 h, and then wash the obtained product with anhydrous ethanol with a volume 9 times that of the product, centrifuge to collect the precipitate, and freeze-dry to obtain the fish scale-derived zinc chelated peptide HAR@Zn.

[0037] Perform Fourier transform infrared spectroscopy on the above-mentioned polypeptide HAR and the fish scale-derived zinc chelated peptide HAR@Zn, and analyze its chelation mechanism.

[0038] As Figure 1 shown in B, after HAR coordinates with Zn2+ to form a chelate, the position of its characteristic absorption peak shifts, and the absorption intensity also changes. The amide I band (1650 cm -1 ) generated by the stretching vibration of C=O significantly decreases in absorption peak after coordinating with Zn2+. Similarly, after coordinating with Zn2+, the absorption peaks at 1550 cm -1 and 1430 cm -1 in HAR weaken in absorption peak intensity and shift to 1540 cm -1 and 1420 cm -1 , indicating that -COO- participates in the covalent binding of Zn2+. Similarly, the absorption peaks (1180 cm -1 and 1130 cm -1)The intensity is significantly weakened, possibly due to the formation of -C-O-Zn complexes. The in-plane vibration of the O═CN bond results in an absorption peak at 721 cm -1 shifting to 722 cm -1 , indicating that Zn2+ coordination may increase the electron cloud density of the oxygen adjacent to C═O in HAR. This series of spectral changes indicates that amino nitrogen, carboxyl oxygen, and carbonyl oxygen are all involved in Zn2+ coordination.

[0039] The performance of the above-mentioned fish scale-derived zinc chelated peptide HAR@Zn was measured, and the results are recorded in Figures 2 - 7 .

[0040] (1) Antibacterial activity of HAR@Zn against Vibrio alginolyticus 2512

[0041] Determination method: The MIC was determined by the microbroth dilution method. The specific operation is as follows: First, add 50 μL of LB broth to a 96-well plate, and then add 50 μL of HAR@Zn to the first column of the 96-well plate and mix well. Gradient dilution was carried out along columns 2 to 10 using the two-fold dilution method, and then 50 μL of the strain 2512 suspension (1×10 5 CFU / mL) was added. Column 11 was used as a blank control (50 μL of medium), and column 12 was used as a negative control (50 μL of medium + 50 μL of sterile water). Incubate at 30 °C for 24 h, and the lowest concentration corresponding to no bacterial growth is the MIC of HAR@Zn.

[0042] The results are as Figure 2 shown, and the MIC of HAR@Zn against Vibrio alginolyticus 2512 is 74.66 μg / mL.

[0043] (2) Anti-biofilm activity of HAR@Zn against Vibrio alginolyticus 2512

[0044] Determination method: It was determined by crystal violet staining. Add cover slips to a 6-well plate, take 200 μL of the activated Vibrio alginolyticus 2512 and culture it in LB broth. After incubating at 30 °C for 12 h, add HAR@Zn and incubate at 30 °C for 24 h. Gently wash each well 3 times with phosphate buffer (PBS, pH = 7.2). Air-dry the adherent cells at 60 °C for 30 min, then stain with 200 μL of 0.1% (w / v) crystal violet at room temperature for 5 min, and gently wash each well again with PBS (pH = 7.4). After staining, dissolve the stain in glacial acetic acid for 10 min. Subsequently, measure the absorbance at 600 nm using a microplate reader and observe with an optical microscope.

[0045] Figure 3 The results show that HAR@Zn not only inhibits the planktonic Vibrio alginolyticus 2512 but also inhibits the biofilm formation of strain 2512.

[0046] (3) Antibacterial spectrum of HAR@Zn against pathogenic bacteria

[0047] Determination method: Using Vibrio alginolyticus 2512, Vibrio parahaemolyticus 2503, Vibrio harveyi 2510, Escherichia coli K88, Pseudomonas aeruginosa PAO1, Listeria monocytogenes 19115, Bacillus cereus 63302, Enterococcus faecium 29122, methicillin-resistant Staphylococcus aureus (MRSA) 43300, and Staphylococcus aureus 6538 as indicator bacteria. Mix the above-mentioned indicator bacteria (1×10 7 CFU / mL) and pour the mixed bacteria onto the plate and inoculate them in LB agar medium. Then use a 6 mm punch to make holes, and add 50 μL of HAES@Zn (50 mg / mL) into the holes. Place it in a biochemical incubator and culture for 24 h. Use the cross method to evaluate the diameter (mm) of the inhibition zone.

[0048] As Figure 4 shown, HAR@Zn has antibacterial activity against the above ten foodborne pathogenic bacteria and its activity is stronger than that of inorganic zinc. Among them, the activity against Escherichia coli K88 (26.94 ± 0.30 mm) is the best, followed by Vibrio parahaemolyticus 2503 (25.74 ± 0.14 mm), Vibrio alginolyticus 2512 (23.99 ± 0.11 mm) and MRSA (23.70 ± 0.49 mm). The activity against Enterococcus faecium 29122 (19.73 ± 0.74 mm) is the worst.

[0049] (4) Hemolytic activity of HAR@Zn on mouse red blood cells

[0050] Determination method: Use the red blood cell lysis method to study the hemolytic activity of HAR@Zn. First, prepare a 4% red blood cell suspension from fresh mouse blood. Add 500 μL of red blood cell suspension into a 1.5 mL centrifuge tube. Then add 500 μL of PBS (negative control), 0.1% Triton X-100 (positive control) and different concentration gradients of HAR@Zn into each centrifuge tube. Then incubate in a 37 °C constant temperature incubator for 4 h. Then centrifuge the mixture, transfer the supernatant to a 96-well plate, and measure the absorbance at OD540.

[0051] Figure 5 The results showed that HAR@Zn had no obvious hemolytic activity on mouse red blood cells.

[0052] (5) Cytotoxicity of HAR@Zn against mouse macrophage RAW264.7

[0053] Determination method: The thiazolyl blue colorimetric method was used to determine the toxicity of HAR@Zn against RAW264.7 cells. The specific steps were as follows: After resuscitating the cryopreserved cells, they were inoculated into the medium (containing 10% fetal bovine serum and 1% double antibody) for subculture (37 °C, 5% CO2); and the concentration was adjusted to 2 - 4×10 5 cells / mL with the medium; 50 μL of the cell suspension and 50 μL of different concentrations of the antibacterial agent were taken and incubated in a 96-well plate for 24 h (37 °C, 5% CO2), and then 25 μL of MTT (5 mg / mL) was added to the 96-well plate and incubated for another 4 h; after the incubation, the supernatant was discarded, and the crystals at the bottom of the wells were dissolved with 100 μL of dimethyl sulfoxide, and the absorbance was measured at 570 nm.

[0054] From Figure 6 it can be seen that at the MIC concentration, HAR@Zn had good biocompatibility with RAW264.7 cells, and the cell survival rate > 80%.

[0055] (6) Stability analysis of HAR@Zn

[0056] Determination method: 1 mg / mL pepsin solution (pH = 2.0) and 1 mg / mL trypsin solution (pH = 7.0) were used to simulate gastric fluid digestion and intestinal fluid digestion respectively. First, a HAR@Zn solution was prepared with ultrapure water, and the pH of the solution was adjusted to 2.0. Then, pepsin solution was added to it so that the mass ratio of pepsin to HAR@Zn in the system was 1:50, and it was placed at 37 °C and 200 rpm for reaction for 2 h, and the pH of the solution was adjusted to 7.0, and trypsin solution was added. At this time, the mass ratio of trypsin to HAR@Zn was 1:25, and the reaction continued at 37 °C and 200 rpm for 2 h. After the reaction, it was placed in a boiling water bath at 100 °C to inactivate the enzyme. Subsequently, the supernatant was taken by centrifugation, and the antibacterial activity was determined by referring to the agar diffusion method.

[0057] Figure 7 The results showed that after in vitro simulated gastrointestinal digestion treatment, the antibacterial activity of HAR@Zn against strain 2512 did not change significantly, and it had excellent stability.

[0058] In summary, the fish scale-derived zinc chelated peptide HAR@Zn of the present invention has excellent biocompatibility and stability, broad-spectrum antibacterial activity, and significant biofilm inhibitory effects. These characteristics make it a potential antibiotic alternative for controlling foodborne pathogenic bacteria and provide a promising application solution for extending the shelf life of aquatic products.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A zinc-chelated peptide derived from fish scales, characterized in that, The amino acid sequence of its polypeptide part is HAR.

2. A preparation method of the fish scale-derived zinc chelated peptide as described in claim 1, characterized in that, It includes the following steps: Mix the aqueous polypeptide solution with the inorganic zinc solution, add an organic solvent for chelation reaction, wash the obtained product, centrifuge to collect the precipitate, and freeze-dry to obtain the fish scale-derived zinc chelated peptide HAR@Zn.

3. The preparation method of the scale-source zinc chelated peptide according to claim 2, wherein, The concentration of the aqueous polypeptide solution is 0.1 - 0.15 g / mL, the concentration of the inorganic zinc solution is 2 mol / L, and the mass ratio of the polypeptide to the inorganic zinc is 0.13 - 0.15:

1.

4. The preparation method of the fish scale-derived zinc chelated peptide according to claim 2, characterized in that, The temperature of the chelation reaction is 30 - 40 °C, and the chelation reaction time is 12 - 16 h.

5. Use of the fish scale-derived zinc chelated peptide as claimed in claim 1 or the fish scale-derived zinc chelated peptide prepared by the preparation method as claimed in any one of claims 2 - 4 in the preparation of antibacterial products.

6. The application according to claim 5, characterized in that, The objects to which the antibacterial product acts include Gram-negative bacteria and Gram-positive bacteria.

7. The application according to claim 6, characterized in that, The Gram-negative bacteria include Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, and Pseudomonas aeruginosa, and the Gram-positive bacteria include Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, and Enterococcus faecalis.

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